Electroporation was first discovered in the 1970s when scientists demonstrated the use of electric fields to create or induce/open pores in cells without causing permanent damage to the cells. This discovery was developed to enable the delivery of genes and various molecules into living cells, tissues and organs. By applying an electric field across cells or tissue element, the permeability of cell membrane is enhanced to enable the entry of genes and molecules into the cell cytoplasm. This phenomenon is transient as the pores reseal upon removal of the applied electric field. Presently, the application of electroporation has expanded into various diverse fields such as molecular biology, cell biology, plant genetics, hybridoma technology, agriculture research, gene therapy and others.
Applications of electroporation involve in-vivo or ex-vivo (in vitro) processes where foreign materials are introduced into living cells, tissues or organs. These living cells include human cells, mammalian cells, plant protoplasts, bacteria, fungi and others. Foreign materials, referred herein as “implant agents”, to be introduced into the living cells can include but not limited to genes, DNA, peptides, proteins and other pharmacological compounds. Under in-vivo procedures, the electroporation process occurs within the living organism. Tweezer and needle electrodes are employed to secure the tissue of interest in place, for example the tumor itself and/or the epidermis encompassing the area of cells to be treated in place, while providing an electric field across the specified region of interest. Cell membranes are transiently made porous by the presence of an applied electric field, thereby allowing the implant agents to enter cells concerned, wherein the implant agents act as modifiers to the cell genome. With the ex vivo procedures, electroporation occurs in an artificially cultured environment, whereby external implant agents are introduced to the living cells. Other electrodes in the form of parallel stainless steel or platinum plates, rods or wires can be utilized to create the electric field across the target cell in ex vivo procedures. Hereinafter, the target media can refer to a living cell, a tissue element or a mixture of implant agents and living cells.
In the field of cancer treatment, for example chemotherapy, conventional methods of absorbing anti-cancer drugs by a human body are deleterious to the surrounding healthy cells. Typically, the high dosage of drugs used to eliminate the cancer cells tends to destroy a significant percentage of healthy cells. Furthermore, certain promising anticancer drugs, for example Bleomycin, have demonstrated inability to enter the membrane of cancer cells effectively. Therefore, electroporation is an excellent alternative methodology for the research of chemotherapy, known as electrochemotherapy, whereby electric pulses are used to increase the permeability of cancerous cell walls so as to allow higher concentration of anticancer drugs to penetrate the cancer cell cytosol. Application of electrochemotherapy is more beneficial to the patient as it minimizes the dosage of treatment drugs and more importantly, the anticancer drugs are able to enter the cancer cells more effectively with minimal tissue damage.
An electroporation method and apparatus generating and applying an electric field according to a user-specific pulsing scheme is taught in U.S. Pat. No. 5,869,326. FIGS. 5-9 of the U.S. patent illustrates various user-specified pulse shapes limited to “high” and/or “low” output voltages. While the user may be able to specify the range of the output voltage, the shape of pulses is limited to only rectangular waveforms. In order to achieve an effective and reliable electroporation treatment, physical parameters such as duration, strength, form and frequency of pulses must be controllable over the treatment region of interest. Hence it is imperative to achieve an apparatus and method for electroporation that can provide programmable arbitrary waveform pulsing trains for optimizing the treatment condition.
Electroporation allows the insertion of certain implant agents to selectively treat undesirable cells without damaging the surrounding healthy cells or tissues. However, the electric pulses typically used in electroporation may cause considerable discomfort and permanent side effects to patients. Major factors affecting the cell permeability induced by electroporation are dependent on important parameters, for example electric pulse strength, pulse duration, pulse shapes and pulse intervals. These physical parameters must be appropriately configured for each individual treatment, as every patient is unique, to prevent irreversible damage to the target cells due to excessive pulse strength. Ineffective opening of the pores, insufficient pulse strength and other capability limiting parameters will also affect the reliability of the treatment. Therefore, optimization of the electric field strength and pulse duration to maximize cell survival and efficient treatment is of extreme importance, and is difficult to achieve based on traditional and conventional single pulse methodologies. Furthermore, the biological properties, for example the type, size and electric conductivity of the cell, may affect the treatment result. Hence there is a need to devise an apparatus and method for generating, applying and monitoring an optimized electric field according to a user configured arbitrary waveform pulsing train.
The present invention optimizes, monitors, modifies and records all processes parameters and conditions of gene transfection, during electroporation with the provision of advanced performance capabilities and flexibilities to configure all process parameters including but not limited to pulse shape, frequency, duration, field strength and other critical important influencing factors.